UPLC-MS-based urine metabolomics reveals indole-3-lactic acid and phenyllactic acid as conserved biomarkers for alcohol-induced liver disease in the Ppara-null mouse model

Since the development and prognosis of alcohol-induced liver disease (ALD) vary significantly with genetic background, identification of a genetic background-independent noninvasive ALD biomarker would significantly improve screening and diagnosis. This study explored the effect of genetic background on the ALD-associated urinary metabolome using the Ppara-null mouse model on two different backgrounds, C57BL/6 (B6) and 129/SvJ (129S), along with their wild-type counterparts. Reversed-phase gradient UPLC-ESI-QTOF-MS analysis revealed that urinary excretion of a number of metabolites, such as ethylsulfate, 4-hydroxyphenylacetic acid, 4-hydroxyphenylacetic acid sulfate, adipic acid, pimelic acid, xanthurenic acid, and taurine, were background-dependent. Elevation of ethyl-β-d-glucuronide and N-acetylglycine was found to be a common signature of the metabolomic response to alcohol exposure in wild-type as well as in Ppara-null mice of both strains. However, increased excretion of indole-3-lactic acid and phenyllactic acid was found to be a conserved feature exclusively associated with the alcohol-treated Ppara-null mouse on both backgrounds that develop liver pathologies similar to the early stages of human ALD. These markers reflected the biochemical events associated with early stages of ALD pathogenesis. The results suggest that indole-3-lactic acid and phenyllactic acid are potential candidates for conserved and pathology-specific high-throughput noninvasive biomarkers for early stages of ALD.

Effects of buffering properties and undissociated acid concentration on dissolution of dental enamel in relation to pH and acid type

To quantify the relationships between buffering properties and acid erosion and hence improve models of erosive potential of acidic drinks, a pH-stat was used to measure the rate of enamel dissolution in solutions of citric, malic and lactic acids, with pH 2.4-3.6 and with acid concentrations adjusted to give buffer capacities (β) of 2-40 (mmol·l(-1))·pH(-1) for each pH. The corresponding undissociated acid concentrations, [HA], and titratable acidity to pH 5.5 (TA5.5) were calculated. In relation to β, the dissolution rate and the strength of response to β varied with acid type (lactic > malic ≥ citric) and decreased as pH increased. The patterns of variation of the dissolution rate with TA5.5 were qualitatively similar to those for β, except that increasing pH above 2.8 had less effect on dissolution in citric and malic acids and none on dissolution in lactic acid. Variations of the dissolution rate with [HA] showed no systematic dependence on acid type but some dependence on pH. The results suggest that [HA], rather than buffering per se, is a major rate-controlling factor, probably owing to the importance of undissociated acid as a readily diffusible source of H(+) ions in maintaining near-surface dissolution within the softened layer of enamel. TA5.5 was more closely correlated with [HA] than was β, and seems to be the preferred practical measure of buffering. The relationship between [HA] and TA5.5 differs between mono- and polybasic acids, so a separate analysis of products according to predominant acid type could improve multivariate models of erosive potential.

Identification of noninvasive biomarkers for alcohol-induced liver disease using urinary metabolomics and the Ppara-null mouse

Alcohol-induced liver disease (ALD) is a leading cause of nonaccident-related deaths in the United States. Although liver damage caused by ALD is reversible when discovered at the earlier stages, current risk assessment tools are relatively nonspecific. Identification of an early specific signature of ALD would aid in therapeutic intervention and recovery. In this study, the metabolic changes associated with ALD were examined using alcohol-fed male Ppara-null mouse as a model of ALD. Principal components analysis of the mass spectrometry-based urinary metabolic profile showed that alcohol-treated wild-type and Ppara-null mice could be distinguished from control animals without information on history of alcohol consumption. The urinary excretion of ethyl-sulfate, ethyl-beta-d-glucuronide, 4-hydroxyphenylacetic acid, and 4-hydroxyphenylacetic acid sulfate was elevated and that of the 2-hydroxyphenylacetic acid, adipic acid, and pimelic acid was depleted during alcohol treatment in both wild-type and the Ppara-null mice albeit to different extents. However, indole-3-lactic acid was exclusively elevated by alcohol exposure in Ppara-null mice. The elevation of indole-3-lactic acid is mechanistically related to the molecular events associated with development of ALD in alcohol-treated Ppara-null mice. This study demonstrated the ability of a metabolomics approach to identify early, noninvasive biomarkers of ALD pathogenesis in Ppara-null mouse model.

Regulation and structure of YahD, a copper-inducible / serine hydrolase of Lactococcus lactis IL1403

Lactococcus lactis IL1403 is a lactic acid bacterium that is used widely for food fermentation. Copper homeostasis in this organism chiefly involves copper secretion by the CopA copper ATPase. This enzyme is under the control of the CopR transcriptional regulator. CopR not only controls its own expression and that of CopA, but also that of an additional three operons and two monocistronic genes. One of the genes under the control of CopR, yahD, encodes an α/β-hydrolase. YahD expression was induced by copper and cadmium, but not by other metals or oxidative or nitrosative stress. The three-dimensional structure of YahD was determined by X-ray crystallography to a resolution of 1.88 Å. The protein was found to adopt an α/β-hydrolase fold with the characteristic Ser-His-Asp catalytic triad. Functional testing of YahD for a wide range of substrates for esterases, lipases, epoxide hydrolases, phospholipases, amidases and proteases was, however, unsuccessful. A copper-inducible serine hydrolase has not been described previously and YahD appears to be a new functional member of this enzyme family.

Coordinated gene expression in adipose tissue and liver differs between cows with high or low NEFA concentrations in early lactation

Dairy cows with high and low plasma non-esterified fatty acid (NEFA) concentrations in early lactation were compared for plasma parameters and mRNA expression of genes in liver and subcutaneous adipose tissue. The study involved 16 multiparous dairy cows with a plasma NEFA concentration of >500 mumol/l [n = 8, high NEFA (HNEFA)] and <140 mumol/l [n = 8, low NEFA (LNEFA)] in the first week post-partum (pp). Blood samples, adipose and liver tissues were collected on day 1 (+1d) and at week 3 pp (+3wk). Blood plasma was assayed for concentrations of metabolites and hormones. Subcutaneous adipose and liver tissues were analysed for mRNA abundance by real-time qRT-PCR encoding parameters related to lipid metabolism. Results showed that mean daily milk yield and milk fat quantity were higher in HNEFA than in LNEFA cows (p < 0.01), and the NEB was more negative in HNEFA than in LNEFA in +3wk too (p < 0.05). HNEFA cows had slightly lower (p < 0.1) insulin concentrations than LNEFA cows across the study period, and the body condition score decreased more from +1d to +3wk in HNEFA than in LNEFA (p = 0.09). The mRNA abundance of genes in the liver related to fatty acid oxidation (carnitine palmitoyltransferase 2 and very long chain acyl-coenzyme A dehydrogenase) and ketogenesis (3-hydroxy-3-methylglutaryl-coenzyme A synthase 2) were lower in HNEFA than in LNEFA cows. No differences between the two groups were observed for mRNA expression of genes in adipose tissue. The number of calculated significant correlation coefficients (moderately strong) between parameters in the liver and in adipose tissue was nearly similar on +1d, and higher for HNEFA compared with LNEFA cows in +3wk. In conclusion, dairy cows with high compared with low plasma NEFA concentrations in early lactation show differentially synchronized mRNA expression of genes in adipose tissue and liver in +3wk that suggests a different orchestrated homeorhetic regulation of lipid metabolism.

Icatibant , the bradykinin B2 receptor antagonist with target to the interconnected kinin systems

INTRODUCTION: HOE-140/ Icatibant is a selective, competitive antagonist to bradykinin (BK) against its binding to the kinin B2 receptor. Substitution of five non-proteogeneic amino acid analogues makes icatibant resistant to degradation by metalloproteases of kinin catabolism. Icatibant has clinical applications in inflammatory and vascular leakage conditions caused by an acute (non-controlled) production of kinins and their accumulation at the endothelium B2 receptor. The clinical manifestation of vascular leakage, called angioedema (AE), is characterized by edematous attacks of subcutaneous and submucosal tissues, which can cause painful intestinal consequences, and life-threatening complications if affecting the larynx. Icatibant is registered for the treatment of acute attacks of the hereditary BK-mediated AE, i.e., AE due to C1 inhibitor deficiency. AREAS COVERED: This review discusses emerging knowledge on the kinin system: kinin pharmacological properties, biochemical characteristics of the contact phase and kinin catabolism proteases. It underlines the responsibility of the kinins in AE initiation and the potency of icatibant to inhibit AE formation by kinin-receptor interactions. EXPERT OPINION: Icatibant antagonist properties protect BK-mediated AE patients against severe attacks, and could be developed for use in inflammatory conditions. More studies are required to confirm whether or not prolonged and frequent applications of icatibant could result in the impairment of the cardioprotective effect of BK.

Proteome and radioimmunoassay analyses of pituitary hormones and proteins in response to feed restriction of dairy cows

The hypothalamic-pituitary system controls homeostasis during feed energy reduction. In order to examine which pituitary proteins and hormone variants are potentially associated with metabolic adaptation, pituitary glands from ad libitum and energy restrictively fed dairy cows were characterized using RIA and 2-DE followed by MALDI-TOF-MS. We found 64 different spots of regulatory hormones: growth hormone (44), preprolactin (16), luteinizing hormone (LH) (1), thyrotropin (1), proopiomelanocortin (1) and its cleavage product lipotropin (1), but none of these did significantly differ between feeding groups. Quantification of total pituitary LH and prolactin concentrations by RIA confirmed the results obtained by proteome analysis. Also, feed energy restriction provoked increasing non-esterified fatty acid, decreasing prolactin, but unaltered glucose, LH and growth hormone plasma concentrations. Energy restriction decreased the expression of glial fibrillary acidic protein, triosephosphate isomerase, purine-rich element-binding protein A and elongation factor Tu, whereas it increased expression of proline synthetase co-transcribed homolog, peroxiredoxin III, beta-tubulin and annexin A5 which is involved in the hormone secretion process. Our results indicate that in response to feed energy restriction the pituitary reservoir of all posttranslationally modified hormone forms remains constant. Changing plasma hormone concentrations are likely attributed to a regulated releasing process from the gland into the blood.

Genome sequence of Enterococcus hirae (Streptococcus faecalis) ATCC 9790, a model organism for the study of ion transport, bioenergetics, and copper homeostasis

Enterococcus hirae ATCC 9790 is a Gram-positive lactic acid bacterium that has been used in basic research for over 4 decades. Here we report the sequence and annotation of the 2.8-Mb genome of E. hirae and its endemic 29-kb plasmid pTG9790.

Cardiac troponins as indicators of acute myocardial damage in dogs

Cardiac troponin I (cTnI) and T (cTnT) have a high sequence homology across phyla and are sensitive and specific markers of myocardial damage. The purpose of this study was to evaluate the Cardiac Reader, a human point-of-care system for the determination of cTnT and myoglobin, and the Abbott Axsym System for the determination of cTnI and creatine kinase isoenzyme MB (CK-MB) in healthy dogs and in dogs at risk for acute myocardial damage because of gastric dilatation-volvulus (GDV) and blunt chest trauma (BCT). In healthy dogs (n = 56), cTnI was below detection limits (<0.1 microg/L) in 35 of 56 dogs (reference range 0-0.7 microg/L), and cTnT was not measurable (<0.05 ng/mL) in all but 1 dog. At presentation, cTnI, CK-MB, myoglobin, and lactic acid were all significantly higher in dogs with GDV (n = 28) and BCT (n = 8) than in control dogs (P < .001), but cTnT was significantly higher only in dogs with BCT (P = .033). Increased cTnI or cTnT values were found in 26 of 28 (highest values 1.1-369 microg/L) and 16 of 28 dogs (0.1-1.7 ng/mL) with GDV, and in 6 of 8 (2.3-82.4 microg/L) and 3 of 8 dogs (0.1-0.29 ng/mL) with BCT, respectively. In dogs suffering from GDV, cTnI and cTnT increased further within the first 48 hours (P < .001). Increased cardiac troponins suggestive of myocardial damage occurred in 93% of dogs with GDV and 75% with BCT. cTnI appeared more sensitive, but cTnT may be a negative prognostic indicator in GDV. Both systems tested seemed applicable for the measurement of canine cardiac troponins, with the Cardiac Reader particularly suitable for use in emergency settings.

Skin rejuvenation without a scalpel. I. Fillers

Fillers are an important tool in the armamentarium of the physician combating aging phenomena. A wide variety of filler substances are now available that meet many, but by far not all, needs in aesthetic medicine. The most commonly used substances now are hyaluronic acid and collagen preparations that have slightly different indications, but collagen requires pre-use testing to rule out inflammatory complications. Poly-L-lactic acid has gained its place in the filling of adipose tissue wasting in HIV-infected patients. Autologous fat is easy to harvest and inject and has virtually no risk of adverse side effects. Permanent fillers may be of advantage but carry the risk of permanent adverse reactions. Skillful combination of different fillers as well as with botulinum toxin injections and other cosmetic procedures may give optimal results.

Copper induction of lactate oxidase of Lactococcus lactis: a novel metal stress response

Lactococcus lactis IL1403, a lactic acid bacterium widely used for food fermentation, is often exposed to stress conditions. One such condition is exposure to copper, such as in cheese making in copper vats. Copper is an essential micronutrient in prokaryotes and eukaryotes but can be toxic if in excess. Thus, copper homeostatic mechanisms, consisting chiefly of copper transporters and their regulators, have evolved in all organisms to control cytoplasmic copper levels. Using proteomics to identify novel proteins involved in the response of L. lactis IL1403 to copper, cells were exposed to 200 muM copper sulfate for 45 min, followed by resolution of the cytoplasmic fraction by two-dimensional gel electrophoresis. One protein strongly induced by copper was LctO, which was shown to be a NAD-independent lactate oxidase. It catalyzed the conversion of lactate to pyruvate in vivo and in vitro. Copper, cadmium, and silver induced LctO, as shown by real-time quantitative PCR. A copper-regulatory element was identified in the 5' region of the lctO gene and shown to interact with the CopR regulator, encoded by the unlinked copRZA operon. Induction of LctO by copper represents a novel copper stress response, and we suggest that it serves in the scavenging of molecular oxygen.

Ridge augmentation by applying bioresorbable membranes and deproteinized bovine bone mineral: a report of twelve consecutive cases

OBJECTIVE: Lateral ridge augmentations are traditionally performed using autogenous bone grafts to support membranes for guided bone regeneration (GBR). The bone-harvesting procedure, however, is accompanied by considerable patient morbidity. AIM: The aim of the present study was to test whether or not resorbable membranes and bone substitutes will lead to successful horizontal ridge augmentation allowing implant installation under standard conditions. MATERIAL AND METHODS: Twelve patients in need of implant therapy participated in this study. They revealed bone deficits in the areas intended for implant placement. Soft tissue flaps were carefully raised and blocks or particles of deproteinized bovine bone mineral (DBBM) (Bio-Oss) were placed in the defect area. A collagenous membrane (Bio-Gide) was applied to cover the DBBM and was fixed to the surrounding bone using poly-lactic acid pins. The flaps were sutured to allow for healing by primary intention. RESULTS: All sites in the 12 patients healed uneventfully. No flap dehiscences and no exposures of membranes were observed. Nine to 10 months following augmentation surgery, flaps were raised in order to visualize the outcomes of the augmentation. An integration of the DBBM particles into the newly formed bone was consistently observed. Merely on the surface of the new bone, some pieces of the grafting material were only partly integrated into bone. However, these were not encapsulated by connective tissue but rather anchored into the newly regenerated bone. In all of the cases, but one, the bone volume following regeneration was adequate to place implants in a prosthetically ideal position and according to the standard protocol with complete bone coverage of the surface intended for osseointegration. Before the regenerative procedure, the average crestal bone width was 3.2 mm and to 6.9 mm at the time of implant placement. This difference was statistically significant (P<0.05, Wilcoxon's matched pairs signed-rank test). CONCLUSION: After a healing period of 9-10 months, the combination of DBBM and a collagen membrane is an effective treatment option for horizontal bone augmentation before implant placement.

Determination of caries risk at resin composite margins

PURPOSE: To design an artificial mouth in order to evaluate if a new diagnostic tool (Clinpro Cario Diagnosis) can be used for early detection of secondary caries at resin composite margins in vitro. METHODS: 32 intact human third molars received standardized Class-V resin composite restorations (Tetric Ceram bonded with Syntac SC). After storage for 4 weeks at 37 degrees C, teeth were subjected to 5,000 or 10,000 thermocycles (+/- 5 degrees C and +/- 55 degrees C) and polysiloxane impressions were taken. Streptococcus mutans 10449 (SM) was used in a nutrition medium to initiate a secondary caries process. Daily, the teeth were incubated for 2 x 2.5 hours in SM containing nutrition medium followed by 2 x 9.5 hours incubation in artificial saliva. Teeth were investigated after total incubation periods of 4, 6, and 8 weeks. After the different incubation protocols, the restoration margins were evaluated for infection and secondary caries processes in using Clinpro Cario Diagnosis which measures site-specifically the lactic acid production of SM in response to a sucrose challenge. The color signal was read 5 minutes after removal of the diagnostic impression. After thermocycling and biological load cycling, precision polysiloxane impressions were taken and replicas were investigated under a light microscope for gap widths at enamel and dentin margins. Demineralization was evaluated by fluorescence microscopy in using a special FITC filter. The demineralization depths at the cavity margin were calculated with Xpert for Windows using a pixel distance of 5 microm. RESULTS: After the different thermocycling protocols, no differences in gap widths and demineralization depths were found (P > 0.05). After SM incubation, gap widths and demineralization depths were significantly dependent on SM incubation time and previous number of thermocycles (P < 0.05). Lactic acid formations of SM were detectable by Clinpro Cario Diagnosis at dentin cavosurface margins formed after 6 weeks of incubation with SM (P < 0.05).

Detection of type III secretion genes as a general indicator of bacterial virulence

Type III secretion systems of Gram-negative bacteria are specific export machineries for virulence factors which allow their translocation to eukaryotic cells. Since they correlate with bacterial pathogenicity, their presence is used as a general indicator of bacterial virulence. By comparing the genetic relationship of the major type III secretion systems we found the family of genes encoding the inner-membrane channel proteins represented by the Yersinia enterocolitica lcrD (synonym yscV) and its homologous genes from other species an ideal component for establishing a general detection approach for type III secretion systems. Based on the genes of the lcrD family we developed gene probes for Gram-negative human, animal and plant pathogens. The probes comprise lcrD from Y. enterocolitica, sepA from enteropathogenic Escherichia coli, invA from Salmonella typhimurium, mxiA from Shigella sonnei, as well as hrcV from Erwinia amylovora. In addition we included as a control probe the flhA gene from E. coli K-12 to validate our approach. FlhA is part of the flagellar export apparatus which shows a high degree of similarity with type III secretions systems, but is not involved in pathogenicity. The probes were evaluated by screening a series of pathogenic as well as non-pathogenic bacteria. The probes detected type III secretion in pathogens where such systems were either known or were expected to be present, whereas no positive hybridization signals could be found in non-pathogenic Gram-negative bacteria. Gram-positive bacteria were devoid of known type III secretion systems. No interference due to the genetic similarity between the type III secretion system and the flagellar export apparatus was observed. However, potential type III secretion systems could be detected in bacteria where no such systems have been described yet. The presented approach provides therefore a useful tool for the assessment of the virulence potential of bacterial isolates of human, animal and plant origin. Moreover, it is a powerful means for a first safety assessment of poorly characterized strains intended to be used in biotechnological applications.